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. 2008 Dec;25(12):2845-52.
doi: 10.1007/s11095-008-9654-8. Epub 2008 Jul 1.

Microfabricated Particles for Engineered Drug Therapies: Elucidation Into the Mechanisms of Cellular Internalization of PRINT Particles

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Free PMC article

Microfabricated Particles for Engineered Drug Therapies: Elucidation Into the Mechanisms of Cellular Internalization of PRINT Particles

Stephanie E A Gratton et al. Pharm Res. .
Free PMC article

Erratum in

  • Pharm Res. 2009 Feb;26(2):488

Abstract

Purpose: To investigate the cellular internalization pathways of shape- and size-specific particles as a function of zeta potential in different cell types.

Methods: A top-down particle fabrication technique called PRINT was utilized to fabricate monodisperse 1 microm cylindrical particles. Cellular internalization of these PRINT particles was monitored using confocal microscopy, flow cytometry, and transmission electron microscopy. The endocytic pathway used by 1 microm cationic PRINT particles was evaluated using different inhibitory strategies. Cytotoxicity assays were used to determine the toxicity of both cationic and anionic PRINT particles in multiple cell types.

Results: Particle internalization was confirmed using confocal microscopy, flow cytometry and transmission electron microscopy. The mechanism of internalization of positively charged PRINT particles was found to be predominantly clathrin-mediated endocytosis and macropinocytosis with very few particles utilizing a caveolae-mediated endocytic pathway. The exposed charge on the surface of the particles had a significant effect on the rate of endocytosis in all cell types tested, except for the macrophage cells. No significant cytotoxicity was observed for all PRINT particles used in the present study.

Conclusions: Cylindrical 1 microm PRINT particles were readily internalized into HeLa, NIH 3T3, OVCAR-3, MCF-7, and RAW 264.7 cells. Particles with a positive zeta potential exhibited an enhanced rate of endocytosis compared to negatively charged particles with identical sizes and shapes. It was found that PRINT particles with a positive zeta potential were endocytosed into HeLa cells using predominantely clathrin-mediated and macropinocytotic pathways.

Figures

Fig. 1
Fig. 1
Chemical structures of monomers and a partial structure of the PRINT particles.
Fig. 2
Fig. 2
A representative scanning electron micrograph of the 1 μm cylindrical particles used in the present study.
Fig. 3
Fig. 3
Confocal micrographs of 1 μm PRINT particles internalized in HeLa cells after a 4 h incubation period.
Fig. 4
Fig. 4
Transmission electron micrographs of 1 μm PRINT particles internalized in HeLa cells after a 4 h incubation period.
Fig. 5
Fig. 5
Determining the cellular internalization pathway of 1 μm positively charged PRINT particles.
Fig. 6
Fig. 6
Internalization of 1 μm PRINT particles by various cell lines. Cells were dosed with various concentrations of particles for 4 h at 37°C. A Cellular internalization profiles; B cytotoxicity of particles. Red circles represent positively charged particles; blue circles represent negatively charged particles. The data shown were representative of more than three separate experiments.

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